Program Director/Principal Investigator (Last, First, Middle): Swanson, Patrick 3R56 AI055599-06A1W1 ABSTRACT B and T lymphocytes form the foundation of our adaptive immune system and have the unique capacity for antigen-specific recognition mediated by immunoglobulins (Igs) and T cell receptors (TCRs), respectively. The Ig and TCR genes are unique because the exons encoding the variable domains of Igs and TCRs must be assembled from discrete gene segments by somatic DNA rearrangement to gain functionality. This rearrangement process, called V(D)J recombination, is initiated when two proteins, called RAG1 and RAG2, bridge two antigen receptor gene segments through protein-DNA interactions with a recombination signal sequence (RSS) that flanks each gene segment, and then cleaves the DNA to separate the RSS from the gene coding segment (the "cleavage phase"). Subsequently, the four DNA ends are reorganized, processed, and rejoined via the non-homologous end-joining (NHEJ) DNA repair pathway to form a "signal joint" from two RSS ends and a "coding joint" from two coding ends (the "joining phase"). While the basic steps of V(D)J recombination are fairly well understood, many important details remain unclear, particularly where the cleavage and joining phases intersect. For example, some evidence suggests that the RAG proteins play an active role in guiding the transfer of DNA ends to the NHEJ machinery, but a mechanistic understanding of this process has not been fully elucidated. Part of the difficulty in elucidating the interplay between the two phases of V(D)J recombination may be due to the historical use of truncated, catalytically active "core" forms of RAG1 and RAG2 in biochemical assays for these reactions, which may be unable to fully stabilize association with regulatory and NHEJ factors. We have developed strategies to purify full-length RAG proteins that increase their suitability for biochemical analysis, and used these RAG preparations to obtain evidence that full-length RAG1 associates with the Ku70/Ku80 complex involved in NHEJ, possibly through novel interaction partners. To gain additional insight into molecular mechanisms that control and connect the cleavage and joining phases of V(D)J recombination, we propose to: (i) determine how the "non-core" portions of the RAG proteins contribute to post-cleavage complex stability and modulate the composition, enzymatic activity, protein-DNA contacts in discrete RAG complexes assembled on DNA substrates containing a pair of RSSs;(iii) characterize newly identified factors found to interact with full-length RAG1 for their biological activity and physiological importance in V(D)J recombination. Greater knowledge of how the RAG proteins initiate cleavage and guide the repair the RAG-mediated DNA breaks will improve our understanding of the mechanisms contributing to impaired and aberrant V(D)J recombination that is suspected to underlie certain forms of immunodeficiency and lymphoid malignancy, respectively.